Fibroblasts were isolated from skin specimens by enzymatic digestion. Briefly, explants were de-epidermized using a dispase solution (dispase activity 14 U/ml; Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C and then were dissolved into a collagenase III solution (2.4 U/ml; Sigma-Aldrich) for 30 minutes. Fibroblasts obtained were passaged twice and cultured at a density of 1 × 106 cells per flask in DMEM (Sigma-Aldrich) supplemented with penicillin (100 U/ml; Sigma-Aldrich), streptomycin (100 μg/ml; Sigma-Aldrich), amphotericin B (0.25 μg/ml; Sigma-Aldrich), glutamine (2 mM; Sigma-Aldrich), and 10 % FBS (Sigma-Aldrich), followed by incubation at 37 °C in an atmosphere of 5 % CO2 and 95 % air until confluence (1 week) in 75-cm2 flasks (BD Costar, Cambridge, MA, USA). Viability was estimated by trypan blue staining (Sigma-Aldrich). Fibroblasts were used at third passage (P3) for cocultures.
Dispase solution
Manufactured by Merck Group
Sourced in United States
Dispase solution is a cell dissociation enzyme used in cell culture and tissue engineering applications. It is a neutral protease that can effectively and gently dissociate a variety of cell types from their extracellular matrix or tissue.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (2 mentions)
Trypan blue staining, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
α mem medium, by Serva Electrophoresis (1 mentions)
α mem medium, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Gene spring software 11, by Agilent Technologies (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Agilent microarray scanner, by Agilent Technologies (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
7 protocols using dispase solution
1
Fibroblast Isolation and Culture Protocol
2
Conjunctival Epithelial Cell Isolation
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A homogenous conjunctival epithelial cell suspension was established to extract mRNA from isolated conjunctiva-derived cells (day 0). Briefly, disinfected conjunctival tissue was exposed to 1.2 U/mL dispase solution (Sigma-Aldrich) for 2 hours at 37 °C under continuous agitation. The incomplete attached cells were mechanically recovered, using a cell scraper, and the obtained cell suspension was lysed using the RNeasy microkit, according to the manufacturer’s instructions.
3
Isolation and Culture of Skin Cells
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Adult skin tissue samples were obtained from donors undergoing elective abdominal dermolipectomy (age 25–58 years, BMI <30, n = 77) with no objection certificate according to the bioethic law no. 2004–800 of August 6, 2004. Keratinocytes were isolated after epidermis and dermis digestion with 10 mg/ml dispase solution (Sigma Aldrich) in Hank’s Balanced Salt Solution (HBSS, ThermoFisher) then trypsine solution (Life technologies) to dissociate epidermis after filtration (100 µm, EMD Millipore). Keratinocytes were cultivated in Dermalife K medium (Cell System) supplemented with 1% ASP. Skin fibroblasts were obtained using dermis explants cultivated in α-MEM medium (Life technologies) with 1% ASP and 20% FBS. Adipose Stromal Cells (ASC) were obtained by digestion of subcutaneous adipose tissue with 0,4 U/ml collagenase NB4 (SERVA electrophoresis) solution in α-MEM medium then removal of floating adipocytes to isolate and culture the stromal vascular fraction in α-MEM medium with 1% ASP and 10% FBS. Cells were used from passage 0 to 4 (P0 to P4).
4
Pemphigus Vulgaris IgG-Induced Keratinocyte Dissociation
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HaCaT cells were cultivated in 24-well plates with 600,000 cells per well in DMEM + GlutaMAX (Gibco, Grand Island, NY, USA) containing 1 mM CaCl2 in a humidified and controlled atmosphere (5% CO2) at 37°C. Twenty-four hours after reaching confluency, positive control AK23 (10 µg/ml), HD IgG (62.5 µg/ml), PV IgG (62.5 µg/ml), or IgG-depleted fractions collected from IgG-specific affinity purification (62.5 µg/ml) were added and incubated for 24 h. The amount of purified IgG used for the keratinocyte dissociation test of PV patients in CR whose sera contained only the anti-Dsg3 IgG4 subclass (before and after treatment) was adjusted to the level of anti-DSG3 Abs measured in the sera collected at baseline from the corresponding patients by a cross multiplication. Subsequently, the cells were treated with dispase solution (2.4 U/ml; Sigma-Aldrich, St. Louis, MO, USA) at 37°C until monolayers were released from plates. Monolayers were stained with crystal violet (Sigma-Aldrich) and subjected to mechanical stress by vigorously pipetting 7 times with a 1-ml pipette. Cell fragments were fixed, photos were taken from each well, and cell fragments were counted manually. All experiments were performed in triplicate.
5
Differential Expression of microRNAs in Keloid Skin
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Strips of keloid and normal skin tissues were incubated in 2.5 mg/ml dispase solution (Sigma-Aldrich, St Louis, MO) at 4 °C for 16 hours, and the epidermis and dermis were separated. Total RNA of epidermis were extracted using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX) and RNA integration was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples from 3 keloids and 3 normal skin with the RNA integrity number (RIN) ≥ 6.0 and 28S/18S ratio ≥0.7 were detected using Agilent’s Human miRNA Microarray Release 18.0 (containing 1887 human microRNAs, Agilent Technologies) at Shanghai Biochip Co., Ltd., China. The slides were scanned by Agilent Microarray Scanner with Feature Extraction Software 10.7, and raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (all by Agilent Technologies). Differentially expressed microRNAs were identified by significance analysis of microarrays (SAM, http://www-stat.stanford.edu/tibs/SAM/index.html ). MiRNA expression was considered down- or up-regulated if the fold change in expression level (Keloid/Normal skin) was <−2 or >2 and a false discovery rate (FDR) corrected p-value was <0.05.
6
Keratinocyte Dissociation Assay for Pemphigus Vulgaris
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A keratinocyte dissociation assay is currently the main tool for the analysis of antibody-induced acantholysis in PV in vitro [26 (link)], and it was performed as previously described in [24 (link)]. In brief, HaCaT cells were cultivated in 24-well plates with DMEM containing GlutaMAX (Gibco, Grand Island, NY, USA) and CaCl2 (1 mM) in a controlled atmosphere (CO2 5%) at 37 °C. Twenty-four hours after reaching the confluence, positive control (AK23: 10 µg/mL), purified IgG from HD (62.5 µg/mL) or from PV patients (62.5 µg/mL) were added and incubated for 24 h. The HaCaT cells were then treated with a dispase solution (2.4 U/mL; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C until the monolayers were separated from the plate. The monolayers were stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) and mechanical stressed by pipetting 7 times with a 1 mL pipette. After the fixation of cell fragments, pictures were taken, and the number of fragments were counted manually. Each experiment was performed in triplicate.
7
Extraction of ECM-Rich Dermal Proteins
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All the animal protocols received approval from the Institutional Animal Care and Use Committee of our institution. Full-thickness skin was harvested from Sprague Dawley rats (250-350 g) immediately following euthanasia by intracardial injection of potassium chloride. Subcutaneous fat and epidermis were carefully dissected away from the dermis. ECM-rich extracts were isolated from the dermis samples for gelation, as described previously. [5, 6] Briefly, the dermis samples were cut into small sections (1-2 mm in thickness) and suspended in dispase solution (Sigma, St. Louis, MO, USA; 2 ml/g of tissue) at 4°C for 15 min. The tissues were then rubbed over a cell sieve before homogenization in a high salt buffer containing protease inhibitors [0.05 M Tris pH 7.4, 3.4 M sodium chloride (NaCl), 4 mM of ethylenediaminetetraacetic acid, 2 mM of N-ethylmaleimide, 0.001 mg/ml pepstatin, 0.01 mg/ml aprotonin, 0.001 mg/ml leupeptin, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (Sigma)]. The tissue was then incubated in 2 M urea buffer (0.15 M sodium chloride, 0.05 Tris pH 7.4; 1 ml/g of tissue) overnight at 4°C. The mixture was centrifuged at 14,000 g for 20 min and the supernatant containing the extracted ECM proteins was stored at 4°C until further use.
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